Temperature-programmed capillary electrophoresis for the analysis of high-melting point mutants in thalassemias

Electrophoresis
C GelfiS Fattore

Abstract

The behavior of different sieving polymers for unambiguous determination of point mutations in genomic DNA, based on electrophoresis in thin capillaries, is evaluated. High melters from thalassemia patients are separated by exploiting the principle of denaturing gradient gel electrophoresis, in fact, of its variant utilizing temperature gradients (TGGE), along the migration path, encompassing the melting points of both homo- and heteroduplex, polymerase chain reaction (PCR)-amplified DNA fragments. Unlike TGGE, where the temperature gradient exists along the separation space, the denaturing temperature gradient in the fused-silica capillaries is time-programmed, so as to reach the Tm's of all species under analysis prior to electrophoretic transport past the detector window. The DNA fragments are injected in a capillary maintained (by combined chemical and thermal means) just below the expected Tm values. The deltaT applied is rather minute (1-1.5 degrees C) and the temperature gradient quite shallow (e.g., 0.05 degrees C/min). The denaturing thermal gradient is generated internally, via Joule heat produced by voltage ramps. This method is applied to the analysis of the most common point mutations in thalassemias, characterized...Continue Reading

References

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Citations

Feb 12, 1998·Electrophoresis·P G Righetti, C Gelfi
Oct 25, 2000·Journal of Chromatography. a·A BossiA P Turner
Nov 22, 1997·Trends in Biotechnology·K R MitchelsonL J Kricka
Jun 17, 1998·Forensic Science International : Synergy·P G Righetti, C Gelfi
Dec 8, 2019·Journal of Clinical Medicine·Eitan Fibach
Nov 2, 2007·Chemical Reviews·Karel Kleparník, Petr Bocek

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